Acanthamoeba myosins IA and IB are unusual in that they consist of only a single, relatively small, heavy chain and one light chain and are unable to form the bipolar filaments typical of other myosins and thought to be necessary for their function. Yet myosins IA and IB have very high actin-activated ATPase activities and are able to crosslink actin filaments into gels and cause superprecipitation that is dependent on ATP hydrolysis. From the unusual triphasic pattern (activation, inhibition and then reactivation) of their ATPase activities as a function of actin concentration, and their ability to cross-link F-actin, we proposed that myosins IA and IB contain two actin binding sites - one insensitive to the presence of ATP and unrelated to ATPase activity and an ATP-sensitive binding site that is associated with catalytic activity. This hypothesis has received several kinds of direct experimental support. (1) Myosin bridges of about 7 nm, the hydrodynamic diameter of single molecules, have been directly visualized in negatively stained electron microscopic images of actomyosin IB complexes. (2) Cross-linking actin filaments by enzymatically inert cross-linking proteins or shortening the filaments by addition of gelsolin had the predicted results of facilitating and inhibiting, respectively, the cooperative interaction of myosin I molecyles on the actin filaments. (3) The myosin IA heavy chain has been proteolytically cleaved into two peptides of 30,000 and 100,000 daltons. The C-terminal 30,000-dalton peptide binds to actin (but does not cross-link filaments, with the same low Kd in the presence or absence of ATP and has no catalytic activity. The 100,000-dalton peptide has the associated light chain and the regulatory phosphorylation site. It binds to F-actin much more tightly in the absence than in the presence of ATP and, when phosphorylated, has full actin-activated ATPase activity but not the cooperative kinetics of the native molecule. The 30,000-dalton peptide contain contains 20% proline and 30% glycine.